Butanol is an aliphatic saturated alcohol with molecular formula of C4H9OH used as a chemical feedstock and as an alternative fuel (Biotechnology and Bioengineering 2008, 101:209-228). As a bulk chemical, butanol is an important precursor of butyl glycol ether, butyl acetate, plasticizer as well butyl acrylate and methacrylate esters used in coating, enamels and lacquers. Besides, it is used in textile industry and applied as a solvent of a wide variety of chemicals (Environmental Technology 2013, 34: 1691-1710).
In terms of energy, butanol is a promising biofuel or biofuel additive. Compared to ethanol, butanol is less hygroscopic and less corrosive. Furthermore, it has a lower vapor pressure, higher energy content and can be blended with gasoline at higher concentrations than ethanol (Journal of Industrial Microbiology and Biotechnology 2012, 39:401-407).
Currently most of the marketed butanol is produced from petrochemical routes. However, butanol can also be produced from biological processes using Clostridium bacteria, which ferments carbohydrates into acetone, butanol and ethanol, called ABE fermentation (Biotechnology progress 2006, 22:673-680).
Clostridium is one of the largest bacterial genera and represents a heterogeneous taxonomic grouping with more than 150 species described (Practical handbook of microbiology. CRC Press, 2008) sharing a number of features as: Gram-positive staining cell wall; anaerobic metabolism; rod-shape morphology; formation of endospores; low GC content; and inability to reduce sulfate (An Introduction to the Family Clostridiaceae. In The Prokaryotes. 4th Volume., 2006). The Clostridium genus is widely spread being found in different habitats as soil, aquatic sediment and gastrointestinal tract (Encyclopedia of microbiology. Elsevier Inc, 2009). Many species have medical importance due to ability to produce powerful toxins and to cause severe diseases. Moreover, many individual species have a high biotechnological potential as the ability to ferment different substrates into organic acids and solvents (Clostridia. Biotechnology Set. 2nd Edition, 2008.
Fermentation production of acetone, butanol and ethanol (ABE fermentation) using Clostridium species was one of the largest and well-established industrial fermentation processes early in 20th century. ABE fermentation started during World War I, when acetone demand increased drastically for the manufacture of munitions. During this time, butanol was an unwanted by-product of the fermentation, however afterwards butanol requirement increased and its production became one of largest industrial fermentation processes (Current Opinion in Biotechnology 2011, 22:634-647). Nevertheless, by 1960s fermentative production of butanol had lost its competitiveness due to high cost of raw material also used in animal feed and due to fast growing of the petrochemical industry, which produced butanol in a cheaper way than biological process. (Applied microbiology and biotechnology 2000, 54:162-167).
Recent progress in biotechnology field combined with increasing demand for the use of renewable sources and the high crude oil price have renewed the interest in butanol fermentation for use as chemical and biofuel (Bioresource Technology 2008, 99:5915-5922). However, for the restoration of ABE fermentation as an economically feasible industrial process some challenges must be overcome as the high cost of substrate, the low butanol yield, the low productivity and the high cost of product recovery (Journal of Industrial Microbiology and Biotechnology 2012, 39:401-407). Therefore, in order to improve the fermentation process different approaches are being developed as the use of alternative substrates, optimization of the fermentation process and metabolic engineering of Clostridium species (Current Opinion in Biotechnology 2011, 22:634-647).
Lignocellulose is the most abundant and cheapest renewable source and it does not compete with food products used in animal and human nutrition. Furthermore, this substrate is recognized as having great potential as a carbon source in fermentation (Journal of Industrial Microbiology and Biotechnology 2012, 39:401-407; Current Opinion in Biotechnology 2011, 22:331-336).
However, to be used as a substrate the lignocellulosic material should be processed and hydrolysed in order to break its complex structures, majority cellulose and hemicellulose, into fermentable sugars, like hexoses and/or pentoses, preferably glucose and/or xylose (Applied microbiology and biotechnology 2000, 54:162-167). Hydrolysate is liquid or solid material resultant from the pre-treatment and/or hydrolysis process of the lignocellulosic material. Besides high sugar concentration, as glucose, xylose and arabinose, the hydrolysate may comprise a wide range of toxic compounds, including weak acids, furans and phenolic compounds that can inhibit the fermentative microbes metabolism, and may become a possible barrier in the use of such material in the solvents and biofuels process (Journal of Industrial Microbiology and Biotechnology 2012, 39:401-407).
Clostridia are able to metabolize simple and complex carbon chains such as pentoses, hexoses, CO2 and CO (Bioresource Technology 2012b, 123: 653-663), confirming the potential use of several types of sugars present in lignocellulosic material. Many studies describe that the Clostridium fermentation using lignocellulosic material has not a high solvent yield and productivity (Biotechnology progress 2006, 22:673-680; Bioprocess and Biosystems Engineering 2007, 30:419-427; Biomass and Bioenergy 2010, 34: 559-565; Biomass and Bioenergy 2010, 34: 566-571), however, this fact is not related to the use of lignocellulosic material. The state of the art for other substrates different than hydrolysate also is limited to low solvent yield and productivity, which can be explained by the accumulation of produced solvents and toxic compounds that causes the inhibition of growth and fermentation.
Butanol is the most toxic solvent for Clostridia. Even low concentrations of butanol (7-13 g L−1) can be responsible for almost 50% of bacteria metabolism inhibition considering its growth rate and substrate consumption when compared to the Clostridium growing in a culture media without solvent or toxic products (Biotechnology and bioengineering 2008, 101:209-228).
This explanation supports one of most difficult challenges in the industrial ABE fermentation: the low yield and low productivity of solvents due to the toxicity of solvents to the Clostridium, which has its metabolism interrupted by lower concentration of solvents or any other type of toxic compounds comprised in the culture media, resulting in low concentration of produced solvents or fuels (Trends in biotechnology 1995, 13:259-264; Biotechnology and bioengineering 2008, 101:209-228).
Therefore, in order to produce large amount of solvents or biofuels, some current processes continuously recover produced solvents, leaving a culture media less toxic for Clostridium, which would allow higher growth rate, and consequently higher yield and solvent and/or biofuel productivity, when compared with the process without the recovering step. Otherwise, the solvent recovery step makes the process become more expensive (Food and Bioproducts Processing 2000, 78:139-144) and, consequently, less industrially competitive.
Many strategies using metabolic engineering and adaptive evolution are also being developed to improve the strain tolerance (Food and Bioproducts Processing 2000, 78:139-144). However, these strategies have shown to be expensive, laborious, time-consuming and frequently the increase of solvent production can be achieved only by using alternative culture conditions or fermentation procedures.
In this context, fermentation using immobilized cells has shown one of the most promising solutions to achieve higher cell density and then greater productivities and yields. Additionally, many studies have already demonstrated the potential of this technology to obtain especially high butanol productivities.
Friedl et al. (Biotechnology and Bioengineering 1991, 38:518-527) have studied an ABE fermentation integrated with product removal by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar coupled with pervaporation process. The packed bed consists of a column of packed bonechar where cells are immobilized and through which the substrate flows. However, during the fermentation, a substantial amount of CO2 is produced by the fermentative microorganism and, considering that the packed bed has no flexibility, there is a risk of disrupting the matrix since there is not enough space for the released CO2. A solvent productivity of 3.5 g h−1 and a solvent yield of 0.39 g g−1 were achieved using these apparatus.
Huang et al. (Biotechnology for Fuels and Chemicals 2004, 115:887-898) reported a butanol productivity of 4.6 g L−1 h−1 and a yield of 0.42 g g−1 at a dilution rate of 0.9 h−1 from continuous culture of Clostridium acetobutylicum cultivated in a fibrous bed bioreactor. This apparatus comprises a wound cotton matrix where Clostridium cells are immobilized and it is supported by a stainless steel mesh into the bioreactor. The open spaces between wound layers of the matrix provide the scape of CO2, which was a limitation of Friedl's (1991) technology. However, this technology requires relatively high liquid flow into the bioreactor to reduce limitations of the mass transport through the matrix layers.
Chen et al. (Biotechnology and Bioprocess Engineering 2013, 18:234-241) immobilized Clostridia cells in a pretreated cotton towel and reported an increasing of 28.3% of butanol yield using immobilized cells when compared with a method with cell suspension. To immobilize the Clostridia cells by adsorption, the cotton towels had to be pretreated as described forward: boiled in water, dried in an oven, soaked in a polyethyleneimine solution with pH regulation, washed in distilled water, soaked in glutaraldehyde, prepared with phosphate-buffer saline (PBS), washed with distilled water, dried in oven and stored. This pretreatment is necessary to the cotton towel surface become much rougher and easier to adsorb the cell. The method described by Chen suggested that immobilization process is a possible method to improve butanol yield and productivity due to a probable increased tolerance to butanol, however, the immobilization method has shown complicated and much laborious when compared to the method described in the present invention.
US2013/0211143A1 discloses an apparatus for producing organic solvents and alcohols that comprises a cell-retaining cellulosic matrix in form of sheet, mat or strip to microbe immobilization and consequently for save their biological activities for at least 14 days. The apparatus comprises a chromatography column (bio-column) filled with water, saturated cellulosic fibers and supported by a plastic net. The spruce cellulosic fibers are rolled together into a tubular form with a plastic net and inserted into column. The column is sterilized overnight using ethanol. Actively growing and producing Clostridia cell mass is loaded into the bio-column by pumping cell suspension with a high flow rate through the matrix. After the bio-matrix was saturated with cells the loading stops and the substrate solution feeding is initiated from the separate substrate bottle. The bio-column is part of a complex amount of devices for controlling growth conditions of microbe cells, fermentation, for recovering the solution that comprises the organic solvents, and for recovering the microbe cells.
CN102952745B discloses a method and an apparatus for butanol production comprising a series of successively tank fermentation. The first tank is a producing acid-immobilized reactor, the second one is a butanol-immobilized reactor and the third one is a product collection tank. The fixed reactor could be a packed bed, a fluidized bed or fiber and the immobilization medium consists of agricultural straw after treatment with activated carbon, fiber and any of a corncob.
CN87103534A discloses a method for acetone and butanol production using immobilized Clostridium acetobutylicum and a starch material. The cells are immobilized on a porcelain ring and the method shows efficient performance and allows continuous fermentation. WO 1981001012 A1 discloses a method for production of solvents by immobilized non-growing cells of Clostridium. The cells can be enclosed in a polymeric material or adsorbed in a solid material or chemically bound in a solid carrier and requires the addiction of butyric acid to increase the yield of butanol.
It is important to consider that all the current cell immobilization technologies present a physical retention of the cells support matrix into an apparatus, facilitating separation of the cells from their products, which are toxic for them and reduces its metabolism, consequently reducing solvents and fuels yield. The current technologies also allow smaller bioreactor volumes due to high productivity and minimum nutrient depletion and product inhibition (Microbiological Reviews 1986, 50:484-524).
Another import disadvantage regarding to cell immobilization into a matrix is the gas production during the fermentation which leads to the accumulation of bubbles into the apparatus making the matrix floats and, consequently, taking the immobilized cell out the culture media. All these issues can affect the mill economic competitiveness because of the difficulty and high cost of maintenance, sterilization and replacement of immobilized bioreactor at large scale.
On the other hand, a second strategy that is described in the state of the art to improve butanol production is based on the quorum sensing system. This system allows the bacterium communication and synchronizes some physiological processes on a population scale by synchronizing gene expression (Annu Rev Cell Dev Biol. 2005; 21:319-46; Annu Rev Genet. 2009; 43:197-222).
The quorum sensing mechanism is mediated by extracellular signaling molecules, the autoinducers, whose concentration increases according to bacteria population density. Autoinducers are produced and released by quorum sensing bacteria, which are able to notice a critical threshold concentration of these molecules in the culture medium and modify the global gene expression. Gram-negative bacteria usually use acyl-homoserine lactone (AHL), while Gram-positive bacteria mostly use oligopeptides as the signaling molecules (Applied Microbiology and Biotehnology 2010, 87: 913-923). Usually, each bacterial species produces and responds to an exclusive autoinducer signal. In general, in Gram-negative bacteria, the autoinducers are specific specie and constituted by a variation of the homoserine lactone core. However, in Gram-positive bacteria these molecules are not variations of a single core, each species produces a peptide signal with a unique sequence. Moreover the quorum sensing receptors in Gram-positive bacteria are histidine kinase proteins with low homology in their transmembrane ligand, which defining their specificity (Annu Rev Genet. 2009, 43: 197-222). Although the inducing molecules are unique for each species, quorum sensing system also allows interspecies cell-to-cell communication and even the prokaryotes and eukaryotes communication. Many studies described the quorum sensing inducer, named AI-2, related to interspecies communication, since both Gram-positiva and Gram-negative bacteria sense and respond to this molecule (Current Opinion in Microbiology 2003, 6: 191-197). Other studies reported the production of molecules by plants that appear to mimic the activities of autoinducers and affect some bacteria behaviors regulated by quorum sensing. Among the species of higher plants reported for producing substances analogues to AHL autoinducers are pea, crown vetch, rice, soybean, tomato and Medicago truncatula (The American Phytophatological Society 2003, 16: 827-834).
Although the main focus of quorum sensing studies are defense mechanism and pathogenicity of bacteria, some researchers describe the role of this system during the fermentative metabolism emphasizing its importance for the production of chemicals. Houdt et al. (FEMS Microbiology Reviews 2007, 4: 407-424) reported that butanediol fermentation is regulated by AHL-mediated quorum sensing in Aeromonas hydrophila AH-1N, since disruption of AHL production by gene knockout blocked the bacteria growth and butanediol production. Moreover, the addition of synthetic AHL similar to the one produced by A. hydrophila can restore the butanediol fermentation.
Kosaka et al. (Bioscience, Biotechnology and Biochemistry 2007, 71:58-68) characterized the sol operon, which contains the genes related to solvents production, in Clostridium saccharoperbutylacetonicum N1-4 and suggested that its transcription, and consequently the solvent production, could be regulated by quorum sensing mechanism. A C. saccharoperbutylacetonicum degenerated strain restored its ability to produce solvents when it was cultivated with a fraction of the supernatant recovered from a wild-type culture thereby suggesting the presence of autoinducers in the supernatant and the probably regulation of sol operon by quorum sensing-like signal.
US 2015/0031102 A1 discloses a method for increasing the amount of butanol produced by Clostridium spp. The method comprises the identification of quorum sensing autoinducers in Gram-positive bacteria and their use in a culture medium to improve butanol production. The ethanol production by Zymomonas mobilis is up regulated in the presence of autoinducers, i.e. molecules related to quorum sensing. Based on this result, the U.S. Pat. No. 8,163,526 B2 discloses a method for increasing the production of ethanol by Zymomonas spp. using autoinducer-2 molecules.
Even though quorum sensing shows up as a promising technology for increased fuel and biochemical production by Clostridium, it is still a complex technology to be applied in a large scale. When it comes to Gram-positive bacteria, which quorum sensing occurs mainly by the action of oligopeptides, the main problem regards the fact that these oligopeptides act specifically from species to species and have formed by small combination of amino acids, making them too difficult to be identified by bioinformatic methodology of gene prediction.
In view of the aforementioned needs in the art, improvements are clearly required to manage and increase the solvent production during the fermentation of lignocellulosic biomass by Clostridium species.